Fucose containing proteoglycan or acidic glycan and their pharmaceutical use

ABSTRACT

A class of proteoglycans containing fucosylated acidic glycans, e.g., as produced by marine sponges and sea urchin embryos, have been found to stimulate selective proliferation of mammalian natural killer (NK) cells and γδT cells. These compounds are useful as pharmaceuticals, particularly as immunostimulants, e.g., in the treatment of cancer and viral infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-In-Part Application of U.S. patentapplication Ser. No. 09/731,092, Filed: Dec. 6, 2000. entitled “FucoseContaining Proteoglycan or Acid Glycan and Their Pharmaceutical Use”which was a Continuation-In-Part Application of U.S. patent applicationSer. No. 08/704,777, filed Dec. 13, 1996, entitled “Fucose ContainingProteoglycan or Acid Glycan and Their Pharmaceutical Use” which is anational phase application under U.S.C. § 371 of PCT/IB95/00208 filedMar. 24, 1995 which claims priority of United Kingdom Application No. GB94-05 846.8 filed Mar. 24, 1994, the disclosures of which is herebyincorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a class of proteoglycans having fucosylatedacidic glycan side chains bound to a protein backbone which have beenfound to stimulate selectively proliferation of natural killer (NK)cells and/or γδT cells. They are useful as immunostimulants, e.g., inthe treatment of cancer and viral infections.

2. Description of the Related Art

The proteoglycans of the invention are produced by proliferating cells,for example by sponge cells, sea urchin cells, and, in the case ofhigher animals (including humans), by embryonic cells and tumor cells.In the natural proteoglycan form, the compounds are large (ca. 5000 to30,000 kD) extracellular or membrane-bound molecules having a proteinbackbone which is glycosylated with acidic glycan chains having anunusual polysaccharide sequence containing internal fucose. Thestructure of the acidic glycan side chains of the proteoglycan isolatedfrom the marine sponge Microciona prolifera has been partiallycharacterized (Spillmann, et al., J. Biol. Chem. (1993) 268:13378-13387, contents incorporated herein by reference), and we havepreviously shown that this proteoglycan is involved in cellularaggregation (Misevic, et al., J. Biol. Chem. (1987) 262: 5870-5877;Misevic, et al., J. Biol. Chem. (1990) 265: 20577-20584; Misevic, etal., J. Biol. Chem. (1993) 268: 4922-4929, contents of all of thesearticles incorporated herein by reference). The previously undescribedprotein backbone of the Microciona prolifera proteoglycan has now beenisolated and characterized, and novel proteoglycans derived from spongesof other genera have also been characterized, as described below.

Those concerned with these and other problems recognize the need for animproved fucose containing proteoglycan or acidic glycan and theirpharmaceutical use.

BRIEF SUMMARY OF THE INVENTION

It has now surprisingly been discovered that these compounds are potentstimulators of NK cells and γδT cells. In particular, compounds of theinvention isolated from an organism of all the phyla and preferably:

-   -   from organisms of the Phylum Porifera e.g., of the class        Demospongiae, especially of the order Poecilosclerida, family        Microcionidae (e.g., of the genus Microciona), or family        Mycalidae (e.g., of the genus Mycale), or the order        Halichondrida, family Halichondridae (e.g., of the genus        Halichondria), or the order Hadromerida, family Clionidae (e.g.,        of the genus Cliona), or the order Haplosclerida, family        Haliclonidae (e.g., of the genus Haliclona),    -   and/or from organisms of the phylum Echinodermata.

These compounds have been shown to stimulate selectively differentclones of NK cells and T cells. Moreover, it has been found thatcompounds of the invention have significant anticancer, especiallyantimetastatic, effects in vivo. It is believed that these anticancereffects are due to stimulation in vivo of NK cells and/or γδT cells. Theprecise mechanism of this stimulation is unclear, but without intendingto be bound by a particular theory, we suggest that these cells may bestimulated by polyvalent interactions with fucosylated acidic glycans ofthe class described herein and in this way can identify and destroyhyperproliferating cells expressing similar glycan structures. In apathogenic case, where the hyperproliferating cells are not destroyed inthis manner, it is believed that although the hyperproliferating cellsproduce these acidic glycans, they shed them or present them inmonovalent form or other nonstimulatory or inhibitory form, therebyevading detection and destruction by NK cells and/or γδT cells specificfor such acidic glycans. Application of the compounds of the inventionstimulates NK cells and/or γδT cells specific for such cancer cells,thereby leading to their destruction. Additionally, the compounds of theinvention are useful for stimulating NK cells and/or γδT cells againstviral or retroviral infections. Finally, in monovalent form, thecompounds of the invention are useful for inhibiting the activation ofNK cells and/or γδT cells, thereby finding utility asimmunosuppressants.

The compounds of the invention are selective in their action, in thatparticular compounds of the invention stimulate only particular clonesor subpopulations of NK cells or γδT cells. No significant stimulationof B cells or γδT cells is observed, so undesirable immunostimulation,e.g., an allergenic or autoimmune response, is avoided. Despite thisselectivity, all humans tested, from a variety of ethnic and racialgroups, have cell populations capable of being significantly stimulatedby the compounds of the invention. Compounds having the glycanstructures of the class described herein are found in a wide variety ofhyperproliferating cells from sponges to human tumors, thus the basicstructure of the compounds is highly conserved. It is hypothesized thatcompounds of the class described herein act as signals for stimulatingthe body's defenses against unwanted proliferation of cancerous orinfected cells, and that cancers or resistant viral infections may arisewhen, as described above, these compounds are secreted in nonstimulatoryform. Among the examples described herein, it is noted that compounds ofthe invention isolated from those of the genus Microciona are moreeffective in stimulating NK cells, as described in example 1 below,whereas compounds isolated from the genus Halichondria are moreeffective in stimulating γδT cells, as described in example 9, thusselectivity among cell types receptive to this stimulation is alsopossible.

Other objects, advantages, and novel features of the present inventionwill become apparent from the following detailed description of theinvention when considered in conjunction with the accompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION

The invention thus provides

1. Fucose-containing proteoglycans and acidic glycans, and/orfragment(s) thereof, preferably proteoglycans, e.g., isolated or capableof being isolated from embryonic or neoplastic tissue or from anorganism of all the phyla and preferably from an organism of the phylumPorifera, e.g., as described above, especially of the genera Microcionaand/or Halichondria and/or Mycale and/or Cliona and/or from an organismof the phylum Echinodermata especially of the genus Lytechinus for useas a pharmaceutical or therapeutic agent in vivo or for ex vivo therapy;and pharmaceutical compositions comprising such compounds in combinationwith a pharmaceutically acceptable carrier or diluent.

2. Novel fucose-containing proteoglycans and acidic glycans, and orfragments thereof, preferably proteoglycans, isolated or capable ofbeing isolated from organisms of the genus Halichondria and/or Mycaleand/or Cliona.

3. Novel fucose-containing acidic glycans capable of being isolated froma sea urchin of the genus Lytechinus,

4. A Fucose-containing acidic glycan for use as a pharmaceutical ortherapeutic agent in vivo or for ex vivo therapy; and pharmaceuticalcompositions comprising such compounds in combination with apharmaceutically acceptable carrier or diluent; and capable of bindingto monoclonal antibodies of the type of these named “Block 2” anddescribed in the reference “Misevic; et al., J. Biol. Chem.(1993). 268:4922-4929,

5. A method of stimulating the proliferation of mammalian, e.g., human,NK cells and/or γδT cells comprising contacting said cells with acompound of the invention (a fucose-containing proteoglycan and acidicglycan, and/or fragment thereof, preferably a proteoglycan and/orfragment(s) thereof, e.g., isolated or capable of being isolated fromembryonic or neoplastic tissue or from an organism of the phylumPorifera, or Echinodermata e.g., as described above, especially of thegenera Microciona and/or Halichondria and/or Mycale and/or Cliona,and/or of the phylum Echinodermata especially of the genus Lytechinus,in an ex vivo setting or in vivo, e.g., as a vaccine; or a method oftreating cancer (e.g., preventing or inhibiting onset, growth, ormetastasis of a tumor); or treating or preventing a viral or retroviralinfection, in a mammal, e.g., man; comprising administering apharmaceutically effective amount of a compound of the invention to apatient in need of such treatment; or the use of a compound of theinvention in the manufacture of a medicament for treatment or preventionof cancer or viral or retroviral infections.

6. The use of a fucose-containing proteoglycan or acidic glycan, orfragment thereof, preferably a proteoglycan and/or fragment(s) thereof,e.g., isolated or capable of being isolated from embryonic or neoplastictissue or from an organism of the phylum Porifera and/or Echinodermata,e.g., as described above, especially of the genera Microciona and/orHalichondria, and/or Mycale and/or Cliona for the phylum Porifera,especially of the genus Lyiechinus for the phylum Echinodermata for exvivo stimulation of proliferation of NK cells and/or γδT cells.

7. A method for screening for or detecting an immunosuppressive (e.g.,NK cell and/or γδT cell) inhibitory compound comprising measuringproliferation of NK cells and/or γδT cells in a system containing an NKcell or γδT cell stimulatory concentration of a compound of theinvention in the presence and absence of a test compound; and compoundsidentified using such a method.

8. A gene coding for a protein capable of post-translationalglycosylation to form the proteoglycan of the invention, vectorscontaining such a gene, and transformed cells, especially (i) productioncells, e.g., sponge cells, incorporating such a gene for use inproducing the desired proteoglycan at enhanced levels or (ii) cancercells removed from a patient, transformed with the gene so as to expressthe proteoglycan in stimulatory form, irradiated, and reintroduced intothe patient. The gene for Microciona proteoglycan can be isolated, forexample, using oligonucleotide probes of a cDNA library based on thedisclosed amino acid sequences.

Appropriate dosages of the compounds of the invention will of coursevary, e.g. depending on the condition to be treated (for example thedisease type or the nature of resistance), the effect desired, and themode of administration. In general however satisfactory results areobtained on administration orally, rectally, nasally, topically, orparenterally, e.g. intravenously, for example by i.v. drip or infusion,at dosages on the order of from 0.01 to 2.5 up to 5 mg/kg, e.g. on theorder of from 0.05 or 0.1 up to 1.0 mg/kg. Suitable dosages for patientsare thus on the order of from 0.5 to 125 up to 250 mg i.v., e.g. on theorder of from 2.5 to 50 mg i.v. Pharmaceutical compositions of theinvention may be manufactured in conventional manner, in a suitableaqueous carrier, for example sterile buffered physiological saline.

For ex vivo stimulation of cells, as described more fully in the examplebelow, a suitable amount, e.g., at least 10 ml, of the patient's bloodis removed, peripheral blood mononuclear cells are isolated from theblood, placed in a complete medium in the presence of a stimulatoryconcentration of a compound of the invention, e.g., 10-500 μg/ml, ca.100 μg/ml, optionally in the presence of IL-2, and the culture ismaintained until a significant increase in the population of the desiredcell type is observed, e.g., ca. 2-4 weeks. Following stimulation of thecells, the cells are isolated from the medium, placed in an injectionsolution, e.g., sterile buffered physiological saline or plasma, andinjected back into the patient. The compound of the invention for thisuse can, for example, be a proteoglycan or acidic glycan derived from amarine sponge as described in the examples, but may also be aproteoglycan, acidic glycan or fragment thereof isolated from a cultureof the cancerous cells to be treated.

INDUSTRIAL APPLICATION

The compounds can be useful notably as pharmaceuticals, particularly asimmunostimulants, e.g. in the treatment of cancer and viral infections.

EXAMPLE 1 Preparation of Proteoglycan and Acidic Glycans from Microcionaprolifera

a. Extraction of Proteoglycan from Microciona prolifera

Fresh marine sponges (Microciona prolifera) collected from the Cape Codarea (USA) are rinsed with 0.5M NaCl, 0.18 g/l NaHCO₃, (buffer A) andcut into cubes 1×1 cm. The cubes are incubated in the buffer A (50%suspension) for 12 h at +4□C under gentle rotation. After filtration ofthe sponge cubes suspension through cheese cloth, the cubes were twomore times extracted with the buffer A using the same incubationconditions. The supernatants are either combined or separatelycentrifuged at 3000×g for 30 min at +4□C, and the obtained supernatantis again centrifuged at 12,000×g for 40 min at +4□C. CaCl₂, is added tothe supernatant to a concentration of 20 mM. After 2-12 h gentle shakingat room temperature, the precipitated proteoglycan is centrifuged at3000×g for 20 min at room temperature. The pelleted proteoglycan isdissolved in at lest 20 volumes of 0.5 M NaCl, 2 mM CaCl₂, 20 mM Tris pH7.4 (buffer B) and centrifuged at 10,000×g for 30 min at +4□C to removeinsoluble material. Supernatant was centrifuged at 100,000×g for 4 h at+40 C, and the pelleted proteoglycan redissolved in buffer B atconcentration of 1-2 mg/ml. To the dissolved proteoglycan in buffer Bsolid CsCl₂ is added to make a 50% concentration, and the solution iscentrifuged in a SW rotor at 100,000×g for 36 h at room temperature. Thepelleted proteoglycan is dialyzed against buffer B and stored at +4□C inthe presence of 0.05% NaN₃.

The purified proteoglycan thus obtained exhibits the followingcharacteristics:

-   -   1) Molecular mass: 19,000 kD 0.20%.    -   2) Sedimentation coefficient S_(20w): 58 20%.    -   3) Stability to enzymes: Not digestible with Chondroitinase A,        B, C, Heparinase, Heparitinase, Hyaluronidase and Keratinase.    -   4) Gelation: Forms gel in aqueous salt solution containing more        then 6 mM CaCl₂ or in deionized water.    -   5) Shape determined with atomic force microscopy in liquid and        electron microscopy: circle of 400-500 nm diameter with 10-20        arms 200-300 nm long.    -   6) Stability: circle portion dissociates from arms in aqueous        salt solutions containing less then 1 mM CaCl₂; or in the        presence of EDTA.    -   7) Ca²⁺ binding determined by flame ionization spectrometry:        binds ca. 7000 moles of Ca²⁺/mole of proteoglycan at 2 mM CaCl₂        and ca. 70,000 moles of Ca²⁺/mole of proteoglycan at 20 mM        CaCl₂.    -   8) Dissociation fingerprinting: Dissociation of proteoglycan by        1% SDS at 100□C gave nine fragments ranging from 38-1500 kD on a        5-20% linear gradient polyacrylamide gel after electrophoresis.        These fragments had apparent molecular masses of ca. 1500 kD,        500 kD, 250 kD, 150 kD, 148 kD, 135 kD, 108 kD, 70 kD, and 38        kD. EDTA and heating at 80□C produced fragments of Mr 1500×10³,        250×10³ on gel filtration chromatography. Trypsin digestion        produced fragments of Mr 124×10³, 70×10³, 27×10, 10×10³ gel        filtration chromatography.

Table I shows approximate amino acid (measured by HPLC pico-tag) andapproximate total sugar composition (measured by gas chromatographyafter methanolysis, reacetylation and silylation):

This proteoglycan consists of approximatively 36% by weight proteins and64% by weight carbohydrates. TABLE I Intact proteoglycan (PG) (mol %)Isolated glycans (mol %) mol amino acid/mol PG mol amino acid/ molglycan Asx 12,736 13.4 1.2 33.4 Thr 8,196 8.6 0.6 16.7 Ser 6,179 6.7 0.38.4 Glx 11,475 12.0 0.7 19.5 Pro 5,611 6.0 0.0 0.0 Gly 12,484 13.1 0.513.8 Ala 9,205 9.7 0.1 2.8 Val 5,296 5.8 0.0 0.0 Met 693 0.8 0.0 0.0 Ile3,287 4.5 0.0 0.0 Leu 6,997 7.4 0.1 2.7 Tyr 3,972 4.2 0.0 0.0 Phe 3,5303.7 0.1 2.7 His 945 1.0 0.0 0.0 Lys 1,261 1.3 0.0 0.0 Arg 1,765 1.8 0.00.0 Total 94,629 100% 3.6 100% mol carbohydrate/mol PG mol carbohydrate/mol glycan Fuc 15,069 33.9 9.9 34.7 GlcUA 4,602 10.3 2.0 7.2 Man 4,0359.1 2.7 9.6 Gal 10,907 24.5 7.4 26.1 GlcNAc 9,836 22.2 6.3 22.3 Total44,449 100.0 28.3 100 mol/mol PG SO₄ ²⁻ 8,241Standard deviation is less then 20% of each value. Asx signifies Asn orAsp; Glx signifies Glu or Gln. It is also noted that apparant amounts ofIle and Leu are somewhat variable depending on the preparation. Theamount of uronic acid determined colormetrically is usually 2 timeshigher then the amount determined by gas chromatography. SO₄ ²⁻ wasdetermined by HPLC ion chromatography after hydrolysis of PG.

The N-terminal sequence of the backbone of the molecule is as follows:Seq. I Pro-Leu-Phe-Thr-Val-Pro-Ile-Tyr-Val-Pro- Glu-Asp-Gln-Leu Seq. IIPro-Glu-Val-Gly-Val-Pro-Ile-Tyr-Val-Pro- Glu-Asp-GIn-Leu Seq. IIIPro-Val-Val-Gly-Val-Pro-Ile-Tyr-Val-Pro-Glu- Asp-GIn-Leu. preferablySequence 1.Seq. I-Seq. III correspond to SEQ ID NOS 1-3 respectively.

Trypsin digestion of the molecule provides peptides having thesequences: Seq. IV Phe-Val-Val-Met-Arg Seq. V Pro-Gln-Asp-Pro-Phe Seq.VI Leu-Ala-Gly-Val-Val-Ile Seq. VII Pro-GIn-Ala-Ser-Ser-Gly Seq. VIIIAla-Ala-Gln-Trp-Ile-Gly-Gln-LysSeq. IV-Seq. VIII correspond to SEQ ID NOS 4-8 respectively.b. Isolation of Acidic Glycans from the Microciona proliferaProteoglycan Frozen proteoglycan as obtained above is extracted withwater/methanol/chloroform 3/8/4 V/V/V, and the nonlipid fraction, waspelleted by centrifugation at 4000×g for 15 min at +4° C. Thisextraction is repeated and the pellet is dried under a vacuum. Thepellet is wetted in ethanol and resuspended in 0.1 M Tris pH 8, 1 mMCaCl₂ and 100-200 μg Pronase (Calbiochem) (preincubated for 30 min at60° C. in 0.1M Tris pH 8, 1 mM CaCl₂ per 1-2 mg dried powder material),and the pellet is digested at 60° C. for three days. Two more equivalentportions of preincubated pronase are added at 24 h intervals. DNAse I isthen added (30 μg) and incubation is continued at 37□C in the presenceof 10 mM MgCl₂. The digested sample is then treated again with pronaseand chromatographed through G-25 Sephadex (Pharmacia) column eluted with10 mM pyridine acetate pH 5, void volume fractions are collected andlyophilized, and the glycans thus obtained are dissolved in 50 mM NaOHin the presence of 1M NaHBO₄ and incubated at 45° C. for 16 h (NaOHtreatment may also be omitted). The glycans are passed through Dowex AG50W-X8 column in H+ form (Bio-Rad) eluted with water, nonbound glycansare immediately neutralized and electrophoresed on a 5-20% or 10-40%linear polyacrylamide gradient gels (Tris/borate-EDTA), and separatedacidic glycans of Mr 200×10³ are eluted from gels. (Optionally, theacidic glycans can be sepatated by gel filtration rather thanelectrophoresis). The isolated acidic glycan molecules are desaltedusing P-2 column (Bio-Rad) eluted with 10 mM pyridine acetate pH 5,lyophilized and stored at −20° C.

The acidic glycan fraction is comprised of two major glycans of apparentmolecular mass determined by gel electrophoresis using glycosaminoglycanstandards of ca. 200 kD and 6 kD. The glycans have the following molarcomposition (expressed as moles of monosaccharide units/mole of glycan),as determined by gas chromatography, as shown in Table II: TABLE II 200kD glycan 6 kD glycan Fuc 680 3 Man 20 2 Gal 180 5 GlcNAc 190 14 GlcUA320 7 Asn 1 1Standard deviation is less then 20% of each value. Per mole ofproteoglycan, there are 20 moles of the 200 kD glycan and 1000 moles ofthe 6 kD glycan. The glycans are not digestible with Chondroitinase A,B, C, Heparinase, Heparitinase, Hyaluronidase or Keratinase. They aresoluble in aqueous solutions and do not form gels in 6 mM CaCl₂, saltsolutions. At higher concentrations, e.g. >1 mg/ml water, they willundergo hydrolysis at room temperature.

After partial acid hydrolysis of isolated glycans fragments werepurified by ion exchange chromatography and high performance liquidchromatography. Methylation analysis, sequential enzymatic and chemicaldegradation, ¹H-NMR spectroscopy, fast atom bombardment-massspectrometry, ion spray mass-spectrometry and MALDI-TOF of nine purifiedfragments showed following oligosaccharide structures: Structure 1     6Pyr< >Galβ1-4GlcNAcβ1-3Fuc     4 is repeated 1000 times per moleproteoglycan. Structure 2 GlcNAcβ1-3Fuc 3 SO₃ is repeated 2000 times permole proteoglycan. Structure 3 Galα1-2Galβ1-4GlcNAcβ1-3Fuc 3 SO₃ isrepeated 2000 times per mole proteoglycan. Structure 4(SO₃)GlcNAc-Fuc-Fuc: Structure 5 Fuc-(SO₃)GlcNAc-Fuc Structure 6GlcNAc-(SO₃)Gal-Fuc Structure 7 Gal-(SO₃)Gal-GlcNAc-Fuc-Fuc Structure 8Gal-(SO₃)Gal-GlcNAc-Fuc-Fuc Structure 9 GlcNAc-Fuc-(SO₃)Gal-Fuc

EXAMPLE 2 Preparation of Proteoglycans and Acidic Glycans fromHalichondria panacea

Extraction of proteoglycan from Hatichondria panicea and isolation ofacidic glycans from Halichondria panicea proteoglycan is performed asdescribed in example 1 for Microciona prolifera. The proteoglycan thusobtained has the following characteristics:

-   -   0.1) Molecular mass: 10,000 kD 20%.    -   2) Sedimentation coefficient of S_(20w) 42 20%.    -   4) Stability to enzymes: Not digestible with Chondroitinase A,        B, C, Heparinase, Heparitinase, Hyaluronidase and Keratinase.    -   5) Gelation: Forms gel in aqueous salt solution containing more        then 6 mM CaCl₂ or in deionized water.

This proteoglycan consists of approximately 79% protein and 21%carbohydrate by weight. It has an approximate amino acid composition (asmeasured by HPLC pico-tag) and approximate total sugar composition (asmeasured by gas chromatography) as shown in Table III: TABLE III Aminoacid composition, and carbohydrate composition Intact proteoglycan (PG)(mol %) Amino acid Asx 9.1 Glx 9.2 Ser 7.0 Gly 9.9 Arg 7.6 Thr 10.2 Ala7.0 Pro 8.2 Tyr 4.6 Val 8.6 Met 2.5 Cys 0.1 Ile 6.0 Leu 5.5 Phe 4.8Total 100% Carbohydrate Fuc 12.5 Xyl 1.9 GlcUA 3.2 GalUA 1.7 Man 16.7Gal 36.2 Glc 13.6 GlcNAc 14.2 Total 100.0 mol/mol PG SO₄ ²⁻ 6,250

Standard deviation is less then 20% of each value. Asx signifies Asn orAsp; Glx signifies Glu or Gin. It is also noted that apparant amounts ofIle and Leu are somewhat variable depending on the preparation. Theamount of uronic acid determined colorimetrically is usually 2 timeshigher then the amount determined by gas chromatography. SO₄ ²⁻ wasdetermined by HPLC ion chromatography after hydrolysis of PG.

Isolation of acidic glycans from this proteoglycan in the mannerdescribed in example I gives seven glycans having apparent molecularmass determined by gel electrophoresis using glycosaminoglycan standardsof ca.>1000 kD, 600 kD, 160 kD, 150 kD, 110 kD, 82. kD, and 50 kD.

After partial acid hydrolysis of isolated glycans fragments werepurified by ion exchange chromatography and high performance liquidchromatography. Methylation analysis, sequential enzymatic and chemicaldegradation, ¹H-NMR spectroscopy, fast atom bombardment-massspectrometry, ion spray mass-spectrometry and MALDI-TOF of eightpurified fragments showed following oligosaccharide structures:structure 10 Pyr(4,6)Gal-Gal-Fuc structure 11 Pyr(4,6)Gal-Gal-Fuc-GlcNAcstructure 12 Pyr(4,6)Gal-Fuc         Gal structure 13Pyr(4,6)Gal-GlcNAc-Fuc         Gal structure 14 Pyr(4,6)Gal-GlcNAc-Fuc               Gal structure 15 Pyr(4,6)Gal-Gal-GlcNAc-Fuc            Gal structure 16 Pyr(4,6)Gal-Gal-GlcNAc-Fuc            Pyr(4,6)Gal structure 17 Pyr(4,6)Gal-Gal-Gal-GlcNAc-Fuc            Pyr(4,6)Gal

EXAMPLE 3 Preparation of Proteoglycans and Acidic Glycans from Mycalelingua

Extraction of proteoglycan from Mycale lingua and isolation of acidicglycans from Mycale lingua proteoglycan is performed as described inexample 1 for Microciona prolifera. The proteoglycan thus obtained hasthe following characteristics:

-   -   1) Molecular mass: 12,000 kD 20%.    -   2). Sedimentation coefficient of S_(20W) 48 20%.    -   4) Stability to enzymes: Not digestible with Chondroitinase A,        B, C, Heparinase, Heparitinase, Hyaluronidase and Keratinase.    -   5) Gelation: Forms gel in aqueous salt solution containing more        then 6 mM CaCl₂; or in deionized water.

This proteoglycan consists of approximately 58% protein and 42%carbohydrate by weight. It has an approximate amino acid composition (asmeasured by HPLC pico-tag) and approximate total sugar composition (asmeasured by gas chromatography) as shown in Table IV: TABLE IV Aminoacid composition and carbohydrate composition Intact proteoglycan (PG)(mol %) amino acid Asx 10.8 Glx 9.6 Ser 6.3 Gly 7.7 Arg 9.5 Thr 10.9 Ala8.0 Pro 7.9 Tyr 0.5 Val 9.0 Met 1.8 Cys 0.2 Ile 6.2 Leu 6.0 Phe 5.6Total 100.0 carbohydrate Fuc 29.7 Xyl 1.0 GlcUA 11.5 GalUA 0.8 Man 11.0Gal 15.3 Glc 16.7 GalNAc 6.3 GlcNAc 7.7 Total 100.0 mol/mol PG SO₄ ²⁻12,000Standard deviation is less then 20% of each value. Asx signifies Asn orAsp; Glx signifies. Glu or Gin. It is also noted that apparant amountsof Ile and Leu are somewhat variable depending on the preparation. Theamount of uronic acid determined colormetrically is usually 2 timeshigher then the amount determined by gas chromatography. SO₄ ²⁻ wasdetermined by HPLC ion chromatography after hydrolysis of PG.

EXAMPLE 4 Preparation of Proteoglycans and Acidic Glycans from Clionacelata

Extraction of two proteoglycans from Cliona celata and isolation-ofacidic glycans from Cliona celata proteoglycans is performed asdescribed in example 1 for Microciona prolifera with the exception thatprecipitation with CaCl₂, could be omitted. Two proteoglycan designatedCPGI (more abundant in the first extraction) and CPG2 (more abundant inthe second extraction) thus obtained has the following characteristics:

-   -   1) Molecular mass: CPGI>20,000 kD 20%; CPG2 6,000 kD.    -   2) Sedimentation coefficient of CPGI S_(20W) 125 20%; CPG2 26        S_(20W) 20%.    -   4) Stability to enzymes: Not digestible with Chondroitinase A,        B, C, Heparinase, Heparitinase, Hyaluronidase and Keratinase.    -   5) Gelation: Both proteglycans form viscous gels in aqueous salt        solution containing more then 0.6 mM CaCl₂; or in deionized        water.

CPGI consists of approximately 26% protein and 74% carbohydrate byweight (determined colorimetrically). CPG2 consists of approximately 32%protein and 68% carbohydrate by weight. They have an approximate aminoacid composition (as measured by HPLC pico-tag) and approximate totalsugar composition (as measured by gas chromatography) as shown in TableV: TABLE V Amino acid composition and carbohydrate composition Intactproteoglycan (CPG1) (CPG2) (mol %) (mol %) amino acid Asx 1.0 7.8 Glx5.6 9.5 Ser 7.1 11.3 Gly 10.6 10.9 Arg 23.6 6.0 Thr 18.1 14.1 Ala 0.77.7 Pro 12.9 10.7 Tyr 8.3 0.7 Val 1.9 6.1 Met 2.4 2.4 Cys 0.3 0.2 Ile1.0 3.9 Leu 1.3 5.1 Phe 0.8 3.6 Lys 4.3 0.1 Total 100.0 carbohydrate Fuc11.0 17.8 Xyl 2.2 2.2 GlcUA 9.4 11.0 GalUA 0.7 1.1 Man 1.2 5.9 Gal 6.812.8 Glc 17.2 18.5 GalNAc 32.5 16.2 GlcNAc 19.0 14.8 Total 100.0 mol/molPG mol/mol PG SO₄ ²⁻ 12,000 6,000Standard deviation is less then 20% of each value. Asx signifies Asn orAsp; Glx signifies Glu or Gin. It is also noted that apparant amounts ofIle and Leu are somewhat variable depending on the preparation. Theamount of uronic acid determined colorimetrically is usually 2 timeshigher then the amount determined by gas chromatography. SO₄ ²⁻ wasdetermined by HPLC ion chromatography after hydrolysis of PG.

Proteoglycans purified from Cliona celata using the same procedure as inExample 1. Calcium chlorid precipitation was preformed duringpurification. M_(r) × 8 10⁶ s_(20,w) (S) 46 Carbohydrate/protein ratio(w/w) 36/64

TABLE VB Aminoacid composition (mol %) Asx 7.8 Glx 9.5 Ser 11.3 Gly 10.9Arg 6.0 Thr 14.1 Ala 7.7 Pro 10.7 Tyr 0.7 Val 6.1 Met 2.4 Cys 0.2 Ile3.9 Leu 5.1 Phe 3.6 Lys 0.1 Carbohydrate composition (mol %) Ara 9.4 Fuc22.9 Xyl 1.5 GlcUA 6.4 GalUA N.D. Man 12.9 Gal 11.3 Glc 4.6 GalNAc 9.2GlcNAc 22.0 SO₄ ²⁻(mol/mol) 7000Standard deviation is less then 20% of each value. Asx signifies Asn orAsp; Glx signifies Glu or Gin. It is also noted that apparant amounts ofIle and Leu are somewhat variable depending on the preparation. Theamount of uronic acid determined calorimetrically is usually 2 timeshigher then the amount determined by gas chromatography. SO₄ ²⁻ wasdetermined by HPLC ion chromatography after hydrolysis of PG.N.D.=Not Detected.

After partial acid hydrolysis of isolated glycans fragments werepurified by ion exchange chromatography and high performance liquidchromatography. Methylation analysis, sequential enzymatic and chemicaldegradation, ¹H-NMR spectroscopy, fast atom bombardment-massspectrometry, ion spray mass-spectrometry and MALDI-TOF of 16 purifiedfragments showed following oligosaccharide structures: structure 18Gal-GlcNAc-Fuc(SO₃)-GlcNAc-Fuc structure 19Gal-GlcNAc-Ara(SO₃)-GlcNAc-Fuc structure 20Gal-GalNAc-Fuc(SO₃)-GlcNAc-Fuc structure 21Gal-GalNAc-Fuc(SO₃)-GalNAc-Fuc structure 22Gal-GlcNAc-Fuc(SO₃)-GalNAc-Fuc structure 23Gal-GalNAc-Ara(SO₃)-GlcNAc-Fuc structure 24Gal-GalNAc-Ara(SO₃)-GalNAc-Fuc structure 25Gal-GlcNAc-Ara(SO₃)-GalNAc-Fuc structure 26GlcNAc-Fuc(SO₃)-GlcNAc-Fuc-Fuc structure 27GlcNAc-Ara(SO₃)-GlcNAc-Fuc-Fuc structure 28GalNAc-Fuc(SO₃)-GlcNAc-Fuc-Fuc structure 29GalNAc-Fuc(SO₃)-GalNAc-Fuc-Fuc structure 30GlcNAc-Fuc(SO₃)-GalNAc-Fuc-Fuc structure 31GalNAc-Ara(SO₃)-GlcNAc-Fuc-Fuc structure 32GalNAc-Ara(SO₃)-GalNAc-Fuc-Fuc structure 33GlcNAc-Ara(SO₃)-GalNAc-Fuc-Fuc

EXAMPLE 5 Preparation of Acidic Glycans from Lytechinus pictius

Lytechinus pictus sea urchin eggs and/or embryos (from 2 cell stage toplutes stage) were washed with sterile sea water and pelleted embryoswere extracted with water/methanol/chloroform. 3/8/4 V/V/V, and thenonlipid fraction was pelleted by centrifugation at 4000×g for 15 min at+4° C. This extraction is repeated and the pellet is dried under avacuum. The pellet is wetted in ethanol and resuspended in 0.1 M Tris pH8, 1 mM CaCl₂; and 100-200 μg Pronase (Calbiochem) (preincubated for 30min. at 60° C. in 0.1M Tris pH 8, 1 mM CaCl₂, per 1-2 mg dried powdermaterial), and the pellet is digested at 60° C. for three days. Two moreequivalent portions of preincubated pronase are added at 24 h. intervalsDNAse I is then added (30 μg) and incubation is continued at 37° C. inthe presence of 10 mM MgCl₂. The digested sample is then treated againwith pronase and chromatographed through G-25 Sephadex (Pharmacia)column eluted with 10 mM pyridine acetate pH 5, void volume fractionsare collected and lyophilized, and the glycans thus obtained aredissolved in 50 mM NaOH in the presence of 1M NaHBO₄ and incubated at45° C. for 16 h (NaOH treatment may also be omitted). The glycans arepassed through Dowex AG 50W-X8 column in H+ form (Bio-Rad) eluted withwater, nonbound glycans are immediately neutralized and electrophoresedon a 5-20% or 10-40% linear polyacrylamide gradient gels(Tris/borate-EDTA), and separated acidic glycans of Mr 200×10′ areeluted from gels. (Optionally, the acidic glycans can be sepatated bygel filtration rather than electrophoresis). The isolated acidic glycanmolecules are desalted using P-2 column (Bio-Rad) eluted with 10 mMpyridine acetate pH 5, lyophilized and purified by affinitychromatography with the Block 2 monoclonal antibodies of ref Misevic. etal. mentioned above stored at −20° C.

-   -   1) Molecular mass: 580 kD 20%.    -   2) Sedimentation coefficient 8.5 S_(20w) 20%.    -   4) Stability to enzymes: Not digestible with Chondroitinase A,        B, C, Heparinase, Heparitinase, Hyaluronidase and Keratinase.

5) Gelation: self-interacton-oligomerization in aqueous salt solutioncontaining more then 6 mM CaCl₂ or in deionized water. TABLE VI molcarbohydrate/molacidic glycan (mol. %) Fuc 737 25.40 Xyl 108 3.73 Gal 391.34 Glc 12 0.41 GlcUA 786 27.10 GalNAc 506 17.46 GlcNAc 712 24.56 Total2,900 100.0 mol/mol SO₄ ²⁻ 1,600Standard deviation is less then 20% of each value. The amount of uronicacid determined colormetrically is usually 2 times higher then theamount determined by gas chromatoaraphy. SO₄ ²⁻ was determined by HPLCion chromatography after hydrolysis of PG.

EXAMPLE 6 Ex Vivo Stimulation of Human NK Cells Proliferation byMicrociona prolifera Proteoglycan and by Its Acidic Glycans

Human peripheral blood mononuclear cell (PBMC) are isolated from 10 mlof blood by centrifugation on Ficoll gradient (Pharmacia). Stimulationof PBMC proliferation with 100 μg/ml acidic glycans or proteoglycans isperformed in the presence of complete medium (RPM 1640, 5% human ABserum, 2 mM L-Glutamine, 1 mM Na pyruvate, non-essential amino acids and50 μg/ml Kanamycin). After 5 days 5 U/ml of human recombinant IL-2 isadded. One half of medium is changed when it becomes acidic. After 7,14, 21, 28 and 35 days cells were analyzed by FACS using antibodiesagainst following markers: CD3, TCR αβ, TCR γδ, CD4, CD8-T cells; CD 16,CD56-NK cells; CD20-B cell; CD14 monocytes. Results from five differentdonors after 3 weeks. In the PBMC cultures treated with acidic glycans,NK cells population (CD 16 and CD 56 positive) and (CD3, TCR αβ, TCR γδ,CD4, CD8, CD20 and CD 14 negative) increased from 1-5% to 30-80% of thetotal PBMC, whereas untreated controls remained at a level of 1-5% NKcells. Specific stimulation of NK cells proliferation by glycans wasconfirmed by ³H thymidine incorporation only in isolated clones of NKcells and not of αβT cells isolated from the same PBMC cultures.

EXAMPLE 7 Ex-Vivo Stimulation of Human NK Cells Proliferation by Mycalelingua and Cliona celata Proteoglycans and by Their Acidic Glycans wasSimilar to Microciona prolifera Proteoglycan

The data for example 6 above is applicable hereto.

EXAMPLE 8 Ex Vivo Stimulation of Human NK Cells Proliferation byLytechinus pictus Acidic Glycan with 580 kD was Similar to Microcionaprolifera Proteoglycan

The data for example 6 above is applicable hereto.

EXAMPLE 9 Stimulation of Human γδT Cells Proliferation (Ex Vivo) byMicrociona prolifera Proteoglycan, Halichondria panacea Proteoglycanand/or Their Acidic Glycans

Same culturing procedure as described in the previous example shows thatMicrociona prolifera acidic glycans stimulate only one subpopulation ofγδT cells via T cell receptor with an increase from 5% to 20%.Halichondria panicea proteoglycan and its acidic glycans stimulate adifferent population of γδT cells from 5% to 70%. These data areconfirmed by 3H thymidine incorporation in isolated clones stimulated byspecific acidic glycans.

EXAMPLE 10 Anti-Tumorogenic and Anti-Metastatic Activity ofProteoglycans and Their Acidic Glycans from Microciona prolifera (InVivo)

Seven C-57 black mice are injected i.p. with 300 kg proteoglycan oracidic glycans from Microciona prolifera/200 μl 0.2M NaCl, 2 mM CaCl₂,20 mM. Tris pH 7.4/animal, every day for five days. At day five, animalsare injected with 2.5×10⁴ B-16 melanoma cells per animal. Animals areimmunized for five more days with proteoglycan as described above. Theappearance of tumor, tumor growth, survival of animals and appearance ofmetastasis are observed in immunized animals and compared with controlanimals injected with buffer. Control animals which have not receivedproteoglycan all exhibit marked melanoma growth followed by metastasis.Compared to controls, treated animals exhibit a 20% delay in the time ofappearance and 50% reduction in growth of syngenic B16 melanomas, a 12%increase in the total time of survival of all immunized mice (p=0.0044),and complete inhibition of metastasis.

EXAMPLE 11 Anti-Tumorogenic and Anti-Metastatic Activity ofProteoglycans and Their Acidic Glycans from Halichondria panicea, Mycalelingua, Cliona celata and Lytechinus pictus

The proteoglycans or acidic glycans obtained from Halicohondria panicea,Mycale lingua, Cliona celata and Lytechinus pictus were testedseparately in C-57 black mice in accordance with the protocol describedin example 10. The appearance of tumor, tumor growth, survival ofanimals and appearance of metastasis are observed in immunized animalsand compared with control animals injected with buffer. Control animalswhich have not received proteoglycan all exhibit marked melanoma growthfollowed by metastasis. Compared to the controls, the treated animalsexhibited a delay in the time of appearance and a reduction in growth ofsyngenic B16 melanomas, an increase in the survival time and aninhibition of metastasis which was similar to that achieved with theproteoglycan used in example 10.

EXAMPLE 12 Cloning and Expression of Gene for Proteoglicans fromMicrociona prolifera

Proteoglycan (PG) cDNA is isolated from a random-primed cDNA librarycreated using poly(A)+RNA from Microciona prolifera cells. This cDNAlibrary is screened using the N-terminal amino acid sequence of PGdescribed in example I above by colony hybridization techniques, i.e.,expressing the library in an expression system, preferably E. coli,lysing the colonies, e.g., on nitrocellulose filters, denaturing theirDNA in situ and fixing it on the filter, hybridizing with labeled,preferably radiolabeled, oligonucleotide probes of at least 30 basepairs having cDNA base sequences corresponding to all or a portion ofthe N-terminal sequence of PG, identifying hybridized colonies, andretrieving the corresponding vectors from the library, using chromosomewalking techniques if necessary to isolate and characterize one or morecDNA fragments containing one or more regions coding for glycosylationsites for N-linked glycans. (Note that the cDNA is repetitive, so it isnot necessary to clone, isolate and characterize the entire sequence).Once the desired portion of cDNA has been isolated, it is expressed in asuitable expression system, preferably a eukaryotic system, mostpreferably a sponge. The PG is isolated from the sponge or from theculture medium of the expression system, e.g., using the proceduresoutlined above.

EXAMPLE 13 Therapeutic Anti-Tumoreognic and Anti-Metastatic Activity ofProteoglycans and Their Acidic Glycans from Microciona prolifera,Halichondria panicea, Mycale lingua, Cliona celata and Lytechinus pictus(In Vivo) Injected in Pharmaceutically Acceptable Diluents and NoTherapeutical Effects when Proteoglycans were Injected inPharmaceutically Not Acceptable Hypotonic or Hypertonic DiluentsContaining Freunds Adjuvant

Forty C-57 black mice were injected with 2.5×10⁴ B-16 melanoma cells inleg per animal. After one day animals were injected i.p. or intra tumorfive times with 150 μg proteoglycans or acidic glycans from Microcionaprolifera (eight animals), Halichondria panicea (eight animals), Mycalelingua (eight animals), Cliona celata (eight animals), and Lytechinuspictus (eight animals)/200 μl 0.188NaCl, 1 mM CaCl₂ 10 mM Tris pH7.4/animal, with first three injections given in interval of 3 days andlast two injections in interval of 7 days. The appearance of tumor,tumor growth, survival of animals and appearance of metastasis werecompared with control not proteoglycan treated animals and animalstreated with pharmaceutically not acceptable diluents:

-   1) hypotonic injection of 200 kg proteoglycans/300 μg water with    complete Freunds adjuvant and-   2) hypertonic injection of 200 μg proteoglycan/300 μl 0.5 M NaCl, 2    mM CaCl₂, 20 mM Tris pH 7.4 or bicarbonate (CSW) with complete    Freunds Adjuvant.    Control animals and animals which received therapy with    pharmaceutically not acceptable diluents of hypotonic and hypertonic    proteoglycan with Complete Freunds adjuvant all exhibit marked    melanoma growth followed by metastasis. In same cases animals    treated with pharmaceutically not acceptable diluents of hypotonic    and hypertonic proteoglycan with Complete Freunds adjuvant were more    ill and had reduced life span under the melanoma bourdon when    compared to controls. Animals treated with proteoglycans in    pharmaceutically acceptable diluents in comparison with the control    not treated animals exhibited a 20%-30% delay in time of tumor    appearance, 50%-80% reduction in growth of syngenic B16 melanoma    tumors, a 12%-20% increase in the total time of survival    (p_(max)=0.004), and absence of visible B16 melanoma metastasis.

EXAMPLE 14 Anti-Tumoreognic and Anti-Metastatic Vaccine Activity ofProteoglycans and Their Acidic Glycans from Microciona prolifer,Halichondria panicea, Mycale lingua, Cliona celata and Lytechinus piclus(In Vivo) Injected in Pharmaceutically Acceptable Diluents and NoTherapeutical Effects when Proteoglycans and their Acidic Glycans wereInjected in Pharmaceutically Not Acceptable Hypotonic or HypertonicDiluents Containing Freunds Adjuvant

Fifty C-57 black mice were injected i.p. or intra tumor five times with150 kg proteoglycans or acidic glycans of Microciona prolifera, (tenanimals) Halichondria panicea, (ten animals), Mycale lingua (tenanimals), Cliona celata (ten animals) and Lytechinus pictus (tenanimals)/200 μl 0.188NaCl, 1 mM CaCl₂ 10 mM Tris pH 7.4/animal, withfirst three injections given in interval of 3 days and last twoinjections in interval of 7 days. One day after last proteoglycaninjection 2.5×10⁴ B-16 melanoma cells were injected in leg per animal.The appearance of tumor, tumor growth, survival of animals andappearance of metastasis were compared with control not proteoglycantreated animals and animals treated with pharmaceutically not acceptablediluents:

-   1) hypotonic injection of 200 kg proteoglycans/300 μg double    distilled water with complete Freunds adjuvant and-   2) hypertonic injection of 200 kg proteoglycan/300 μg 0.5 M NaCl, 2    mM CaCl₂, 20 mM Tris pH 7.4 with complete Freunds Adjuvant.    Control animals and animals which received vaccine with    pharmaceutically not acceptable diluents of hypotonic and hypertonic    proteoglycan with Complete Freunds adjuvant all exhibit marked    melanoma growth followed by metastasis. In same cases animals    treated with pharmaceutically not acceptable diluents of hypotonic    and hypertonic proteoglycan with Complete Freunds adjuvant were more    ill and had reduced life span under the melanoma burden when    compared to controls. Animals treated with proteoglycans vaccine in    pharmaceutically acceptable diluents in comparison with the control    not treated animals exhibited a 20% delay in time of tumor    appearance, 50% reduction in growth of syngenic B16 melanoma tumors,    a 12%-20% increase in the total time of survival (pmax 0.004), and    absence of visible B16 melanoma metastasis.

EXAMPLE 15 Ex Vivo Stimulation of Human NK Cells Proliferation byMicrociona prolifera, Halichondria panicea Mycale lingua, Cliona celata,and Lytechinus pictus Proteoglycans and by Their Acidic Glycans Attachedto Tissue Culture Dishes

Tissue culture dishes were coated separately by adsorption or crosslinking to tissue culture dishes using 0.1 μg of proteoglycan or acidicglycan from Microciona prolifera, Halichondria panicea Mycale lingua,Cliona celata and Lytechinus pictus per 1 cm². After drying period of 30min and UV sterilization dishes were washed three times with sterilephosphate buffered isotonic NaCl solution. Human peripheral bloodmononuclear cells (PBMC) are isolated from 10 ml of blood bycentrifugation on Ficoll gradient (Pharmacia). Isolated human PMBC wereresuspended in 5% homologue human sermn of the same donor in RPMI 1640medium, 2 mM L glutamate, 1 mM Na-pyruvate and supplement ofnon-essential amino acids and 50 μg Kanamicine. Medium with homologueserum was change every 3-5 days. After 7, 14, 21 and 28 days cells wereanalyzed by immunofluorescence microscopy using following markers: CD3,TCR αβ, TCRγδ, CD4, CD8-T cells; CD CD16, CD56-NK cells; CD20 B cells;CD14 Monocytes. Within one week the population of NK (1-5% of the totalcells) started to proliferate to reach 100% after three weeks.

EXAMPLE 16 Killing of Human Tumor Cells Ex Vivo with Human Nk CellsProliferated on Microciona prolifer, Halichondria panicea, Mycalelingua, Cliona celata and Lytechinus pictus Proteoglycans and by TheirAcidic Glycans Attached to Tissue Culture Dishes

Tissue cultured human tumor cells lines were added in 1000 times excessto the number of human NK cells proliferated four weeks on Microcionaprolifera, or Halichondria panicea, or Mycale lingua, or Cliona celataor Lytechinus pictus proteoglycans and/or its acidic glycans attached totissue culture dishes. Massive and continuous killing of tumor cellscoming in contact with NK cells is documented microscopically during theperiod of five weeks co culturing period.

In accordance with publications of the International Union of Pure andApplied Chemistry and International Union of Biochemistry and MolecularBiology, Joint Commission on Biochemical Nomenclature, Nomenclature ofCarbohydrates (Recommendations 1996) published in Carbohydrate ResearchVolume 297 Number 1, Jan. 2, 1997, by Elsevier Science LTD, the linkagepoint of glycan fragments are at their respective reducing end with freealdehyde group, in our example fucose. Via this aldehyde group fragmentcan link to any respective hydroxyl group (glycosydic linkage) of theidentical fragment and/or via inactive carrier to the next identicalfragment to form repetitive linear or branched polymers.

EXAMPLE 17 Ex Vivo Stimulation of Human Nk Cells Proliferation,Anti-Tumorogenic and Anti-Metastatic Vaccine Activity, and TherapeuticAnti-Tumoreognic and Anti-Metastatic Activity by Fragments ofFucose-Containing Acidic Glycans or Fucose-Containing Proteoglycans fromMicrociona prolifer, Halichondria panacea, Mycale lingua, Cliona celataand Lytechinus pictus

Same procedures as described in the previous example show that fragmentsof fucose-containing acidic glycans or fucose-containing proteoglycansfrom Microciona prolifer, Halichondria panicea, Mycale lingua, Clionacelata and Lytechinus pictus have similar ex vivo stimulation of humanNK cells proliferation, anti-tumorogenic and anti-metastatic vaccineactivity, and therapeutic anti-tumoreognic and anti-metastatic activity.

Examples of several fragments structures obtained from fucose-containingacidic glycans, shown above structures 1 to 33, can have differentdegree and form of repetitiveness wherein linkages and points ofattachments of repetitive structures are here schematically shown, e.g.each number in the schema is representing one monosaccharide of thefragment, dash (-) is linkage to the following monosaccharide within thefragment, and arrow (→) is linkage point between two fragments withinthe repetitive structure.

Example for trisaccharide sequence GlcAc-Fuc-(SO₃)Ga→GlcNAc-Fuc-(SO₃)Galis then 1-2-3→1-2-3. Based on this schematics below are presenteddifferent forms of repetitive structures, using this example structure,that can be implemented for each fragment structure of fucose-containingacidic glycan naturally obtained or obtained by chemical cross linkingof the monomer fragment.

Besides this homo-type repetitive structures containing only one type ofthe fragment structure, also any hetero-type repetitive structurecontaining any combination of different types of fragments structures ispossible.

Although only an exemplary embodiment of the invention has beendescribed in detail above, those skilled in the art will readilyappreciate that many modifications are possible without materiallydeparting from the novel teachings and advantages of this invention.Accordingly, all such modifications are intended to be included withinthe scope of this invention as defined in the following claims.

1. A pharmaceutical composition which comprises an active ingredientwhich is a fucose-containing proteoglycan or fucose-containing acidicglycan; said active ingredient being in combination with an inactive,pharmaceutically acceptable carrier, diluent or excipient, excludingdouble distilled water or CSW sea water.
 2. The composition of claim 1wherein said fucose containing proteoglycan or fucose-containing acidicglycan is capable of being isolated from an organism any phyla.
 3. Thecomposition of claim 2 wherein said fucose containing proteoglycan orfucose-containing acidic glycan is capable of being isolated from agenus of marine sponge selected from the group consisting of Microciona,Halichondria, Mycale and Cliona.
 4. A fucose-containing proteoglycan orfucose-containing acidic glycan capable of being isolated from a marinesponge of the genus Mycale.
 5. A pharmaceutical composition whichcomprises a fucose-containing acidic glycan capable of being isolatedfrom a sea urchin of the genus Lytechinus in combination with aninactive, pharmaceutically acceptable carrier, diluent or excipient,excluding double distilled water.
 6. A kit of components for ex vivostimulation of proliferation of mammalian NK cells and/or γδT cellscomprising the composition of claim 1 with an inactive substrate.
 7. Thecomposition of claim 6 wherein said phyla is selected from the groupconsisting of Porifera and Echinodermata.
 8. The composition of claim 1wherein said fucose-containing proteoglycan or fucose-containing acidicglycan comprises carbohydrates including 12.5 to 34.7% mole of fucose.9. A fucose-containing proteoglycan or fucose-containing acidic glycanwherein said fucose-containing proteoglycan or acidic glycan whichincludes an oligosaccharide having a repetitive structure selected fromthe group consisting of repetitive structures 1, 2, and 3; whereinrepetitive structure 1 has the structure:    6Pyr< >Galβ1-4GlcNAcβ1-3Fuc    4    wherein repetitive structure 2 is hasthe    structure: GlcNAcβ1-3Fuc 3SO₃    wherein repetitive structure 3has the    structure: Galα1-2Galβ1-4GlcNAcβ1-3Fuc 3SO₃


10. The composition of claim 1 wherein said composition consistsessentially of fucose-containing proteoglycan or fucose-containingacidic glycan and an inactive, pharmaceutically acceptable carrier,diluent or excipient, excluding double distilled water and sea water orCSW sea water.
 11. A composition comprising an active fragment of thefucose-containing proteoglycan or fucose-containing acidic glycanrecited in claim 1 in combination with an inactive, pharmaceuticallyacceptable carrier, diluent or excipient, wherein said fragment canstimulate selectively the proliferation of mammalian NK cells or γδTcells.
 12. A fucose-containing acidic oligosaccharide fragment from afucose-containing acidic glycan or proteoglycan, said fragmentcomprising at least one structure selected from the group consisting ofthe structures 4 to 33: structure 4 (SO₃)GlcNAc-Fuc-Fuc: structure 5Fuc-(SO₃)GlcNAc-Fuc structure 6 GlcNAc-(SO₃)Gal-Fuc structure 7Gal-(SO₃)Gal-GlcNAc-Fuc-Fuc structure 8 Gal-(SO₃)Gal-GlcNAc-Fuc-Fucstructure 9 GlcNAc-Fuc-(SO₃)Gal-Fuc structure 10 Pyr(4,6)Gal-Gal-Fucstructure 11 Pyr(4,6)Gal-Gal-Fuc-GlcNAc structure 12 Pyr(4,6)Gal-Fuc        Gal structure 13 Pyr(4,6)Gal-GlcNAc-Fuc         Gal structure 14Pyr(4,6)Gal-GlcNAc-Fuc               Gal structure 15Pyr(4,6)Gal-Gal-GlcNAc-Fuc             Gal structure 16Pyr(4,6)Gal-Gal-GlcNAc-Fuc             Pyr(4,6)Gal structure 17Pyr(4,6)Gal-Gal-Gal-GlcNAc-Fuc             Pyr(4,6)Gal structure 18Gal-GlcNAc-Fuc(SO₃)-GlcNAc-Fuc structure 19Gal-GlcNAc-Ara(SO₃)-GlcNAc-Fuc structure 20Gal-GalNAc-Fuc(SO₃)-GlcNAc-Fuc structure 21Gal-GalNAc-Fuc(SO₃)-GalNAc-Fuc structure 22Gal-GlcNAc-Fuc(SO₃)-GalNAc-Fuc structure 23Gal-GalNAc-Ara(SO₃)-GlcNAc-Fuc structure 24Gal-GalNAc-Ara(SO₃)-GalNAc-Fuc structure 25Gal-GlcNAc-Ara(SO₃)-GalNAc-Fuc structure 26GlcNAc-Fuc(SO₃)-GlcNAc-Fuc-Fuc structure 27GlcNAc-Ara(SO₃)-GlcNAc-Fuc-Fuc structure 28GalNAc-Fuc(SO₃)-GlcNAc-Fuc-Fuc structure 29GalNAc-Fuc(SO₃)-GalNAc-Fuc-Fuc structure 30GlcNAc-Fuc(SO₃)-GalNAc-Fuc-Fuc structure 31GalNAc-Ara(SO₃)-GlcNAc-Fuc-Fuc structure 32GalNAc-Ara(SO₃)-GalNAc-Fuc-Fuc structure 33GlcNAc-Ara(SO₃)-GalNAc-Fuc-Fuc


13. A fucose-containing acidic oligosaccharide fragment according toclaim 12 wherein the structure 4 to 33 is repetitive.
 14. Thecomposition of claim 11 comprising at least one fucose-containing acidicoligosaccharidic fragment according to claim
 12. 15. The composition ofclaim 11 comprising at least one fucose-containing acidicoligosaccharidic fragment according to claim 13.